Preliminary study to evaluate the phytochemicals and physiochemical properties in red and black date's vinegar

Abstract Antioxidant activity, total phenolic content (TPC), total flavonoids, carotenoids, pH, and total titratable acidity of red and black date's vinegar were analyzed. The extraction method was designed and optimized for this purpose with respect to the variety and solvent concentrations along with the time of ultrasonication. The results showed that red dates' vinegar has significantly (p < 0.05) higher total phenols (3.38 ± 0.13 mg GAE/ml) and antioxidant activity as compared to black dates' vinegar, which had a higher amount of carotenoids (3.43 ± 0.11 mg/100 ml). Similarly, red dates' vinegar has more flavonoids as compared to commercially available Zhenjiang vinegar. In terms of physiochemical properties, both red and black date's vinegar were not significantly different (p > 0.05). Use of 50% and 80% methanol with 25 min of ultrasonication for extraction seemed more effective. The total phenols, flavonoids, antioxidant activity, carotenoids, and physiochemical analysis of the red and black date's vinegar indicated that vinegar from dates (red or black dates) is a competitive product in the marketplace.

In the assessment of phytochemicals in agricultural products, various techniques available have been introduced prominent among them are the colorimetric assays. For colorimetric analysis to be possible, it is obligatory to have an extract or sample free of solid particles. The established method for the extraction of antioxidant compounds varies in few parameters in terms of solvent utilization. However, the key purpose of the extraction is to retain or extract much of the bioactive elements as possible (Saafi, El Arem, Issaoui, Hammami, & Achour, 2009). Earlier, researchers have reported the influence of various parameters (solvent type, ultrasonication time, percentage, and temperature) in the extraction of antioxidant compounds and phenolic compounds (Alothman, Bhat, & Karim, 2009;Spigno, Tramelli, & De Faveri, 2007). The purpose of this study was to evaluate the phytochemical and physiochemical properties of red and black dates' vinegar produced by solid-state fermentation method. Comparison and correlation analysis of phytochemicals and antioxidants properties were done between various types of dates' vinegar. For this purpose, an extraction technique was designed in which variables (ultrasonication time and type of solvent) were optimized: Lastly, the values acquired from tested vinegar were compared with commercially available vinegar. To the best of our knowledge, this paper is the first to study the various phytochemical and physiochemical properties of red and black date's vinegar.

| Samples
Red and black date's vinegar was provided by Liyang Bask Vinegar Co., Ltd, Hebei, China. Dates were grown and harvested in Xinjiang, Hetian (China). Traditional solid-state fermentation method was used to produce vinegar. The aging time of vinegar was more than 1 year at the time of analysis, while the Zhenjiang vinegar and F I G U R E 1 Extraction process

| Extraction method
Because of dissimilar consistencies of the samples analyzed, it was essential to create an extraction method for the determination of antioxidant activity and total phenols. To establish the extraction system, little modification was made in the method of Chen, Fan, Yue, Wu, and Li (2008) and Gorinstein et al. (1999). Optimization of the utmost persuasive parameters in the extraction system was necessary; the parameters for optimization were a time of ultrasonication extraction (10, 15, and 25 min) and solvent percentage (50%, 80%, and 100%). The choice of superlative extraction parameters was made by taking into consideration the maximum values gained in every assessment plus economy of solvent used and time. The extraction method was displayed in Figure 1.

| Determination of total phenolic content
Folin-Ciocalteu's assay (Singleton, Orthofer, & Lamuela-Raventós, 1999) was used to determine the values of total phenolic content (TPC) through gallic acid as the standard. The mixture of sample (50 μl) solution, 3 ml distilled water, Folin-Ciocalteu's reagent solution (250 μl), and 750 μl of 7% Na 2 CO 3 were mixed well and then incubated at room temperature for 8 min. 950 μl of distilled water was added. Afterward, the mixture was allowed to stand at room temperature for 2 hr and measured at 765 nm with distilled water as a blank. The TPC was measured as gallic acid equivalents (mg GAE/ml), and calibration curve linearity range was 50-1,000 μg/ml (r = 0.99). Chen and Ho (1995) Szollosi and Varga (2002) Heimler, Vignolini, Dini, and Romani (2005)   absorbance was measured at 510 nm against distilled water as blank.

| Determination of total flavonoid content
The results were expressed and calculated as milligrams of Rutin (mg Ru/ml) by the calibration curve of Rutin. 10-1,000 μg/ml (r = 0.99) was the linearity range of the calibration curve. Sanusi and Adebiyi (2009) method was used to determine total carotenoids with slight modifications. Concisely, 0.5 ml of date's vinegar was extracted with 5 ml of ethanolic butylated hydroxyl toluene (ethanol/BHT-100:1, v/w) in triplicate for separation and the discharge of carotenoids. After mixing well, it was kept in a water bath for 5 min at 85°C. Then, for saponification 80% KOH (0.5 ml) was added and vortexed completely before placing it back for 10 min at 85°C in a water bath. 3 ml of cold deionized water was added followed by cooling down the mixture in an ice water bath. 3 ml of n-Hexane was added to mixture before 2,500 × g centrifugation for two layers' separation to 5 min. The yellow colored upper layer was moved and collected. Until the upper layer become colorless, this method was repeated for four times.

| Determination of total carotenoid contents
Hence, in each centrifuge tube, 12 ml of hexane was added and the end volume of every tube was noted. The measurement was done against the hexane as the blank at the wavelengths of both 450 and 503 nm. The results were given as mg/100 ml. The total carotenoids were calculated by Equation (1).

| Statistical analysis
One-way analysis of variance (ANOVA) was used to analyze data by using statistical software Minitab version 17 (Minitab Inc., PA, USA). p ≤ 0.05 was considered to indicate a statistically significant variance. Analyses were conducted in triplicate, and results were reported as mean ± standard deviations.

| Extraction process optimization and solvent used
The criteria picked for optimization of the extraction parameters  (Sultana, Anwar, & Ashraf, 2009). In our case, we used various percentages of methanol as a solvent for the extraction process to evaluate the best percentage of methanol. Sultana et al. (2009) reported that use of methanol as a solvent showed the best outcomes in the TPC determination (12.2 ± 0.28 mg GAE/ml) and TFC assay (8.66 ± 0.21 mg Ru/ml) determination, and they also explained that these values were based on the composition of different medicinal plants, and these types of plants comprise various compounds which offer antioxidant activity having various response and solvent affinity of the particular assay. Our outcomes are also in agreement with the preceding study of Chatha, Anwar, and Manzoor (2006), who stated that maximal extract yield (g/100 g) was gained with aqueous methanol from rice bran.

| Solvent percentage effect
Various studies have proposed that the phenol recovery depends In another study, a researcher showed that alcohol and water mixtures exhibited better recoveries of phenolic contents as compared to a solvent system based on monocomponent (Pinelo, Del Fabbro, Manzocco, Nuñez, & Nicoli, 2005). Similarly, Yilmaz and Toledo (2006) reported that in the grape seed powder ethanol extraction, an enhancement in the extracted phenolic content appeared when they increased the volume of water in the mixture (0%-30%) and phenolic content decreased at high percentages of water. The solvent-to-water ratio of 80:20 proved to be best for obtaining maximum values for most of the parameters analyzed.

| Effect of ultrasonication time
The acoustic cavitations and mechanical effects created in the solvent by the passage of an ultrasound wave permit for best diffusion of the solvent into the sample matrix (Lu & Luthria, 2016;Rostagno, Palma, & Barroso, 2003;Wang, Sun, Cao, Tian, & Li, 2008

| Total phenolic content
Phenolics apply their useful health outcomes majorly by their antioxidant activity (Fang, Yang, & Wu, 2002). These compounds are able of reducing the concentration of oxygen, precluding 1st-chain initiation through scavenging initial radicals, crumbling initial products of oxidation to nonradical sorts, and breaking chains to avoid consistent hydrogen cogitation from substrates (Shahidi & Naczk, 2003).
Phenolic compounds subsidize to the inclusive antioxidant activities of the foods originated from plants.
Total phenolic contents of the extracts from selected dates are shown in Table 1. Dates' vinegar from various extraction solvents varied significantly in their TPC (p < 0.05). The TPC of the red date's vinegar ranged from 1.89 to 3.05 mg GAE/ml, while the TPC of black date's vinegar ranged from 1.04 to 1.50 mg GAE/ml. The results proposed that 80% of methanol provide the maximum yields among the three different concentrations of solvent for extracting total phenols from red and black date's vinegar. This finding agreed well with the study of Ubeda et al. (2011), who found that maximum extraction of total phenols from persimmon vinegar was obtained by using 80% of ethanol. Similarly, one researcher showed the same results, who analyzed methanol and ethanol and their mixes with water (80%) as an extraction solvent for therapeutic plants (Sultana et al., 2009). Though there is not much research conducted on red and black date's vinegar, there are several types of research conducted on date fruits. From the outcomes of the total phenolic content of soft, semisoft, and dry dates, in comparison with semisoft and dry date vinegar, the soft date vinegar had the least total phenolic content (Hafzan, Saw, & Fadzilah, 2017). Mansouri, Embarek, Kokkalou, and Kefalas (2005) and Biglari, AlKarkhi, and Easa (2008) reported that TPC of Algerian and Iranian date palm fruit ranged, respectively, from 2.49 to 8.36 mg GAE/100 g of fresh and from 2.89 to 6.64 mg GAE/100 g of dry weight. These levels are almost similar to our results. In the other way, the study showed by Wu et al.

| Radical DPPH scavenging activity
It is commonly accepted that free radicals formed in the body are partly correlated with the cancers etiology and other chronic ailments. Antioxidants from the diet, able of scavenging free radicals, are capable to decrease the disease risk. Hence, it is imperative to evaluate the radical scavenging effect of antioxidants in the date's vinegar. DPPH formed a violet solution in ethanol.
Lessening of DPPH by antioxidants outcomes in deprivation of absorbance. Therefore, the marks of discoloration of the solution specify the scavenging efficiency of the added elements. Kenta. The outcomes showed that Algerian date palm fruit has a lower level of antioxidant as compared to Tunisia based on the same method. They stated that this variation is strongly related to the type of active compound and also to the polyphenolic content present in each variety (Saafi et al., 2009). These levels of DPPH values were almost similar to our results. The outcomes revealed that dates' vinegar either red or black is also free radical scavenger, mainly of peroxyl radicals, which are main diffusers of the oxidation chain of fat, thereby dismissing the chain reaction (Sakanaka & Ishihara, 2008). Whereas positive significant correlations were noted between TFC and DPPH radical scavengers in the phytochemicals (Table 2).

| Total carotenoid content
Carotenoids are an enormous group of pigments that occur naturally in plants, different microorganisms, and algae. They are known to have a defensive effect on cardiovascular diseases, cancer, and chronic diseases. The chemical composition of carotenoids is generally based on a C40 tetraterpenoid structure with an extended conjugated, centrally located double-bond system, which is linked to color presented, and acts as light-absorbing chromophore (Meléndez-Martínez, Britton, Vicario, & Heredia, 2007 fruits. Results of one study showed that TCC in fresh dates Khasab, Fard, and Khalas were 1.31, 1.39, and 3.03 mg/100 g, respectively (Al-Farsi et al., 2005). The highest total carotenoids in Khalas were predictable, as this variety has a yellow color, whereas the other two varieties are red. While in our study black dates' vinegar showed a higher level of carotenoids as compared to red, the outcomes of one study showed that the carotenoid content of freeze-dried date was 0.22 mg/100 g of dry weight (∼0.18 mg/100 g of fresh weight) (Ben-Amotz & Fishier, 1998). However, this is much lesser than those found in the present study, and this is probably due to the existing variance between the two samples in maturation, variety, analysis condition, and storage. In one study, Hart and Scott (1995) surveyed the carotenoid content of fruits and vegetables frequently consumed in the United Kingdom. In eight fruits, total carotenoid content ranged from 0.017 to 2.263 mg/100 g of fresh weight, being highest in mandarins and lowest in strawberries. Hence, dates' vinegar can be considered a good source of carotenoids compared to the above fruits. Further, negative correlations signify that TCC and TPC were not good radical scavengers between the phytochemicals (Table 2). The negative correlation between TCC and total phenolic content of date vinegar might be due to the existence of nonphenolic compounds in date vinegar that contribute to the total carotenoid property of date vinegar (Hafzan et al., 2017).

| Physicochemical properties: pH, total sugar content, and titratable acidity
The total titratable acidity, pH value, and sugar content of date's vinegar are shown in Table 3 (Hafzan et al., 2017;Sakanaka & Ishihara, 2008) and also according to international legislation.

| Comparison with commercial vinegar
Zhenjiang and persimmon vinegar were picked from the local market to compare them with date's vinegar (  (Hafzan et al., 2017). Further, no significant correlations were noted between the dates and commercially available vinegar (  proposed that dates' vinegar either red or black has beneficial health outcomes and might be a competitive product in the market.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

R E FE R E N C E S
Correlation is significant at p < 0.05.